Visible Detection of Microorganisms

ABSTRACT

Methods of detecting very low levels of targets, such as cells, are provided. In some embodiments, for example, the methods can detect bacteria present in a sample at concentrations less than 25 cells/mL. The method involves detecting nanoparticle aggregation in the absence of the target.

This application is a continuation of U.S. application Ser. No.13/300,148, filed on Nov. 18, 2011 and issuing as U.S. Pat. No.9,851,308 issuing Dec. 26, 2017, which application claims the benefit ofU.S. Provisional Application No. 61/415,498, filed Nov. 19, 2010, whichis incorporated by reference herein in its entirety for any purpose.

BACKGROUND

The presence of even low levels of spoilage or pathogenic microorganismsin various products can be dangerous and even lethal. Therefore,sensitive, rapid, and simple-to-use methods for detecting microorganismsthat can be performed in out-of-the-laboratory settings, and byuntrained persons, are preferred over those requiring sophisticatedtechniques that are laborious, time consuming, or require skilledpersonnel to carry out. For example, the analysis of genetic informationbased on the polymerase chain reaction (PCR), a popular method forsensitive detection of bacteria, requires complicated sample preparationprotocols performed by skilled personnel. Some alternative methods areknown, such as detection of intact bacterial cells using specificantibody-antigen binding, but simple, fast, and sensitive methods areneeded.

Since sol particle immunoassays (SPIA) were introduced, target-mediated(i.e., on-target) aggregation of gold nanoparticles (AuNPs) has beenused to visually detect the occurrence of some biological events or thepresence of small biomolecules such as proteins and DNA. However, usingthe principle of on-target aggregation of AuNPs for visual detection oflarge targets such as microorganisms, especially at extremely lowconcentrations, has proved difficult.

SUMMARY

In some embodiments, methods of determining whether a sample comprises atarget are provided. In some embodiments, the method comprises (a)contacting the sample with a linker, wherein the linker comprises afirst functionality and a plurality of second functionalities, whereinthe first functionality is capable of binding to the target, and whereineach of the plurality of second functionalities is capable of binding toa third functionality; (b) contacting the sample from (a) with aplurality of nanoparticles, wherein each of the plurality ofnanoparticles comprises a third functionality that is capable of bindingto the second functionality; and (c) detecting nanoparticle aggregationin the sample from (b), wherein the absence of nanoparticle aggregationindicates that the sample comprises the target.

In some embodiments, the method comprises (a) contacting the sample witha linker, wherein the linker comprises a first functionality and aplurality of second functionalities, wherein the first functionality iscapable of binding to the target, and wherein each of the plurality ofsecond functionalities is capable of binding to a third functionality;(b) contacting the sample from (a) with a plurality of nanoparticles,wherein each of the plurality of nanoparticles comprises a thirdfunctionality that is capable of binding to the second functionality;and (c) detecting nanoparticle aggregation in the sample from (b),wherein the presence of nanoparticle aggregation indicates that thesample comprises the target. In some embodiments, the linker is presentin an excess concentration relative to the concentration of thenanoparticles.

In some embodiments, the first functionality is selected from an antigenbinding region of an antibody, a ligand, a receptor, a small molecule,and a lectin. In some embodiments, the second functionality is selectedfrom biotin, streptavidin, an antigen, an antibody, a ligand, areceptor, a polyhistidine tag, nickel, an aptamer, an aptamers target,trans-cyclooctene, and tetrazine. In some embodiments, the thirdfunctionality is selected from biotin, streptavidin, an antigen, anantibody, a ligand, a receptor, a polyhistidine tag, nickel, an aptamer,an aptamers target, trans-cyclooctene, and tetrazine. In someembodiments, the second and third functionalities together form abinding pair selected from biotin/streptavidin, ligand/receptor,polyhistidine tag/nickel, aptamer/aptamer target, antibody/antigen, andtrans-cyclooctene/tetrazine.

In some embodiments, the linker is an antibody and the firstfunctionality is the antigen binding region of the antibody. In someembodiments, the second functionality is biotin and the thirdfunctionality is streptavidin.

In some embodiments, the nanoparticles are selected from goldnanoparticles, silver nanoparticles, platinum nanoparticles, magnetitenanoparticles, gold/iron alloy nanoparticles, and latex nanoparticles.In some embodiments, the target is selected from prokaryotic cells,eukaryotic cells, and parasites.

In some embodiments, detecting nanoparticle aggregation comprisesdetermining at least one characteristic selected from sample color,UV-VIS spectrum, UV-VIS peak wavelength, and absorbance. In someembodiments, the at least one characteristic of the sample from (b) iscompared to at least one characteristic of a standard. In someembodiments, the standard is a control reaction comprising the linkerand the plurality of nanoparticles, but not the target. In someembodiments, the standard is a representation of at least onecharacteristic of a control reaction that comprises the linker and theplurality of nanoparticles, but not the target.

In some embodiments, kits for determining whether a sample comprises atarget are provided. In some embodiments, a kit comprises (i) a linker,wherein the linker comprises a first functionality and a plurality ofsecond functionalities, wherein the first functionality is capable ofbinding to the target, and wherein each of the plurality of secondfunctionalities is capable of binding to a nanoparticle; and (ii) aplurality of nanoparticles, wherein each of the plurality ofnanoparticles comprises a third functionality that is capable of bindingto the second functionality.

In some embodiments, the first functionality is selected from an antigenbinding region of an antibody, a ligand, a receptor, a small molecule,and a lectin. In some embodiments, the second functionality is selectedfrom biotin, streptavidin, an antigen, an antibody, a ligand, areceptor, a polyhistidine tag, nickel, an aptamer, an aptamers target,trans-cyclooctene, and tetrazine. In some embodiments, the thirdfunctionality is selected from biotin, streptavidin, an antigen, anantibody, a ligand, a receptor, a polyhistidine tag, nickel, an aptamer,an aptamers target, trans-cyclooctene, and tetrazine. In someembodiments, the second and third functionalities together form abinding pair selected from biotin/streptavidin, ligand/receptor,polyhistidine tag/nickel, aptamer/aptamer target, antibody/antigen, andtrans-cyclooctene/tetrazine. In some embodiments, the nanoparticles areselected from gold nanoparticles, silver nanoparticles, platinumnanoparticles, magnetite nanoparticles, gold/iron alloy nanoparticles,and latex nanoparticles.

In some embodiments, the linker is an antibody and the firstfunctionality is the antigen binding region of the antibody. In someembodiments, the second functionality is biotin and the thirdfunctionality is streptavidin.

In some embodiments, a kit further comprises a standard. In someembodiments, the standard is a representation of at least onecharacteristic of a control reaction that comprises the linker and theplurality of nanoparticles, but not the target. In some embodiments, theat least one characteristic is selected from sample color, UV-VISspectrum, UV-VIS peak wavelength, and absorbance.

BRIEF DESCRIPTION OF THE FIGURES

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawings will be provided by the United States Patent Office uponrequest and payment of the necessary fee.

FIG. 1 shows the results of on-target detection of bacteria usingantibody-conjugated gold nanoparticles (AuNPs), as described in Example2. FIG. 1A shows the color of the reactions containing E. coliconcentrations of 10¹ to 10⁹ CFU/400 μL (tubes 1 to 9, respectively).FIG. 1B shows the control reaction (left tube) and the reactioncontaining 10⁹ CFU/400 μL (right tube) after centrifugation.

FIG. 2 shows the results of on-target detection of bacteria using anAuNP concentration that is 25% of the concentration used in theexperiment shown in FIG. 1, as described in Example 3. FIG. 2A shows thecontrol reaction (right tube) and the reaction containing 10⁹ CFU/400 μL(left tube) using the original concentration of AuNPs aftercentrifugation. FIG. 2B shows the control reaction (right tube) and thereaction containing 10⁹ CFU/400 μL (left tube) using 25% of the originalconcentration of AuNPs after centrifugation.

FIG. 3 shows a schematic of a proposed mechanism for off-target celldetection, as discussed in Example 4. FIG. 3A shows binding of AuNPs tothe cell surface through a biotinylated antibody linker. FIG. 3B showsaggregation of AuNPs following cross-linking of multiple AuNPs throughthe biotinylated antibody.

FIG. 4 shows the results of the off-target detection assay described inExample 4. FIG. 4A shows the color difference between samples containingE. coli (red, right tube) and not containing E. coli (purplish, lefttube) after 15 minutes. FIG. 4B shows the same samples after one hour.FIG. 4C shows the UV-VIS spectra of samples containing 10¹ to 10⁷ CFU/mLE. coli, as described in Example 4. The peak absorbance wavelength foreach trace is indicated by an arrow.

FIG. 5 shows the peak absorbance change and peak wavelength shift in amixture containing biotinylated BSA and streptavidin-coated AuNPs, asdescribed in Example 5.

FIG. 6 shows (A) a plot of Δλmax versus biotinylated BSA concentrationfor mixtures of biotinylated BSA and streptavidin-coated AuNPs atvarious time points; (B) a photograph of the mixtures from (A) at the 3hour time point, with an indication of the regions exemplified in (C);and (C) a schematic showing an exemplary mechanism explaining thephenomena observed at different concentrations of biotinylated BSA; asdescribed in Example 5.

FIG. 7 is a diagram of the method involving pre-incubation with freestreptavidin, as described in Example 5.

FIG. 8 shows a plot of Δλmax versus biotinylated BSA concentration formixtures of biotinylated BSA and streptavidin-coated AuNPs in thepresence of various concentrations of free streptavidin, as described inExample 5. The inset shows photographs of the mixtures in the presenceof 10 μg streptavidin.

FIG. 9 shows a plot of Δλmax versus biotinylated antibody concentrationfor mixtures of biotinylated antibody and streptavidin-coated AuNPs inthe presence of various concentrations of free streptavidin, asdescribed in Example 5.

FIG. 10 shows an exemplary method of detecting microbial contaminationin a sample, as described in Example 6.

FIG. 11 shows photographs of assay reactions in the presence of variousconcentrations of bacteria and various concentrations of biotinylatedantibody, as described in Example 6. The red boxes indicate twoexemplary biotinylated antibody concentrations that provided sensitivedetection of low levels of bacteria.

DETAILED DESCRIPTION

The present invention provides a novel strategy for detection oftargets, such as cells. In this strategy, the aggregation ofnanoparticles does not occur in the presence of the target, but ratherindicates the absence of the target. The method uses a bifunctionallinker that comprises a first functionality that binds to a target, anda second functionality that facilitates aggregation of nanoparticles.When the linker is bound to the target, aggregation of the nanoparticlesdoes not occur to a significant extent. In the absence of target,however, the linker is available to facilitate aggregation of thenanoparticles. Aggregation of the nanoparticles can be detected throughchanges in at least one characteristic of the sample. For example, insome embodiments, aggregation causes the color of the sample changes.Further, the present method is effective for detecting targets that aremuch larger than the nanoparticles, such as cells, and is verysensitive—the method can detect E. coli concentrations of less than 25cells/mL. In addition, the reagents used in the present method can bedesigned such that they provide a desired level of specificity for oneor more targets. That is, in some embodiments, reagents can be designedsuch that the method detects, for example, a particular target (such asa particular strain of bacteria). In some embodiments, reagents can bedesigned such that the method detects, for example, two or more targets(such as two or more strains of bacteria), e.g., by binding to afunctionality that is shared among the two or more targets, or by mixingtogether reagents that are specific for each target to be detected, or acombination of the two methods.

Definitions

The terms “bifunctional linker” and “linker” are used interchangeablyherein to refer to a molecule that comprises a first functionality thatbinds to a target, and a second functionality that is capable of bindingto at least one nanoparticle. In some embodiments, a linker comprises aplurality of second functionalities. In some embodiments, a secondfunctionality is capable of binding to a plurality of nanoparticles.

The term “nanoparticles,” as used herein, refers to particles that areless than 1 μm in diameter and which cause a detectable change in asample when they aggregate. Nanoparticles may be of any shape, includingspherical and rod-shaped, so long as the longest dimension is within thediameter thresholds discussed herein. In some embodiments, ananoparticle is less than 0.5 μm, less than 0.1 μm, less than 50 nm,less than 10 nm, less than 5 nm in diameter. In some embodiments,nanoparticles are functionalized with a moiety (referred to herein as a“third functionality”) that interacts with the second functionality of alinker, described above. The term “nanoparticles” includes nanoparticlesthat are functionalized with such a moiety.

The term “target,” as used herein, refers to an entity that is to bedetected. In some embodiments, a target is greater than 0.1 μm in itslongest dimension. In some embodiments, a target is greater than 0.5 μm,greater than 1 μm, greater than 2 μm, or greater than 5 μm in itslongest dimension.

Exemplary Linkers

The methods described herein use a linker comprising a firstfunctionality that is capable of binding to a selected target, and asecond functionality that is capable of binding to at least onenanoparticle. In some embodiments, a second functionality is capable ofbinding to a plurality of nanoparticles. In some embodiments, the linkercomprises a plurality of second functionalities. In some embodiments,the linker facilitates the aggregation of the nanoparticles. Forexample, in some embodiments, a linker comprising a plurality of secondfunctionalities brings a plurality of nanoparticles in close proximitysuch that they aggregate. In some embodiments, the linker comprises asingle second functionality, wherein the second functionality is capableof binding to a plurality of nanoparticles. In some such embodiments,the second functionality brings a plurality of nanoparticles in closeproximity such that they aggregate.

The first functionality, in some embodiments, is any moiety that iscapable of binding to the target. In some embodiments, multiple firstfunctionalities are capable of binding to a single target. Nonlimitingexemplary first functionalities include antigen binding regions ofantibodies, ligands, receptors, small molecules, and lectins (such asmannose-binding lectins), etc. When the first functionality is anantigen binding region of an antibody, the linker may comprise theentire antibody, or just a portion of the antibody that includes theantigen binding region. In some embodiments, when a first functionalityis an antigen binding region of an antibody, the target comprises theantigen on its surface. In some embodiments, the target comprisesmultiple copies of the antigen on its surface such that a plurality oflinkers can bind to a single target. In some embodiments, when a firstfunctionality is a ligand, the target comprises the receptor on itssurface. In some embodiments a first functionality binds to multipletargets, for example, when multiple different targets comprise the sameor a similar antigen on their surfaces. One skilled in the art canselect a suitable first functionality depending on the particulartarget(s) to be detected.

The second functionality, in some embodiments, is a member of a bindingpair, wherein the nanoparticles comprise the other member of the bindingpair (in some embodiments, a third functionality). Nonlimiting exemplarybinding pairs include biotin/streptavidin, ligand/receptor,polyhistidine tag/nickel, aptamer/aptamer target, antibody/antigen,trans-cyclooctene/tetrazine (see, e.g., Haun et al., Nat. Nanotechnol.5(9): 660-6 (2010)), etc. One skilled in the art can select a suitablesecond functionality and/or third functionality depending on the linker,the nanoparticles, and the intended application.

In some embodiments, the linker comprises a plurality of copies of thesecond functionality. In some embodiments, the linker comprises at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 15, or at least 20 copies of the secondfunctionality.

In some embodiments, a second functionality is capable of binding tomultiple copies of the third functionality. In some embodiments, asecond functionality is able to bind to at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, or at least 10 copiesof the third functionality. In some such embodiments, a linker maycomprise one or just a few copies of the second functionality.

Nonlimiting exemplary linkers may comprise proteins, peptides, nucleicacids, aptamers, small molecules, carbohydrates, polymers, bindingpairs, etc. In some embodiments, one or more portions of the protein,peptide, nucleic acid, aptamer, small molecule, carbohydrate, polymer,etc., acts as the first functionality and/or the second functionality ofthe linker. For example, in some embodiments, a linker may comprise abiotinylated protein, wherein the protein is a ligand for a particularreceptor on the surface of a target cell, and the nanoparticles arefunctionalized with streptavidin. In some such embodiments, the firstfunctionality of the linker is the ligand portion of the protein (whichmay be the entire protein) and the second functionality is biotin. Insome embodiments, a linker is a biotinylated antibody. In some suchembodiments, the antigen binding region is the first functionality andbiotin is the second functionality.

Exemplary Nanoparticles

Nonlimiting exemplary nanoparticles include gold nanoparticles, silvernanoparticles, platinum nanoparticles, magnetite nanoparticles,gold/iron alloy nanoparticles, and latex nanoparticles.

In some embodiments, nanoparticles have an average size of between 1 nmand 1 μm. In some embodiments, the nanoparticles are gold nanoparticles(AuNPs). In some embodiments, the gold nanoparticles have an averagesize of between 1 nm and 100 nm. In some embodiments, gold nanoparticleshave an average size of between 1 nm and 50 nm, between 5 nm and 50 nm,between 5 nm and 30 nm, between 5 nm and 20 nm, or between 10 and 15 nm.

For a particular detection method, nanoparticles are selected that aresmaller than the target to be detected. In some embodiments, the targetis at least 10-fold, at least 20-fold, at least 30-fold, at least50-fold, at least 100-fold, or at least 200-fold larger than thenanoparticles. As a nonlimiting example, in a method of detecting E.coli, which is about 1 to 3 μm in its longest dimension, nanoparticlesmay be selected for detection that are between 10 nm and 20 nm. Oneskilled in the art can select suitably sized nanoparticles depending onthe target to be detected.

In some embodiments, nanoparticles are functionalized with (i.e.,comprise) a member of a binding pair (in some embodiments, referred toas a third functionality). In some embodiments, the nanoparticles arefunctionalized with (i.e., comprise) a member of a binding pair thatbinds to the member of the binding pair comprised in the linker (i.e.,the second functionality). Nonlimiting exemplary binding pairs aredescribed in the “Exemplary Linker” section, above. In some embodiments,nanoparticles are functionalized with (i.e., comprise) streptavidin. Oneskilled in the art can select a suitable binding pair depending on theparticular nanoparticles, linker, and application.

Methods of functionalizing nanoparticles are known in the art. In someembodiments, for example, nanoparticles are functionalized withstreptavidin according to the method described in Example 1. One skilledin the art can select a suitable method of functionalizing nanoparticlesdepending on the particular application.

Exemplary Targets

The methods described herein can be used to detect targets that aregreater than 0.1 μm in their longest dimension. In some embodiments, atarget is greater than 0.2 μm, greater than 0.5 μm, greater than 1 μm,greater than 2 μm, or greater than 5 μm. Nonlimiting exemplary targetsinclude prokaryotic cells (such as bacterial cells), eukaryotic cells(including yeast), parasites, etc. In some embodiments, a target is amicrobial food and/or water contaminant.

Nonlimiting exemplary targets that can be detected using the methodsdescribed herein include E. coli (including E. coli 0157:H7),Staphylococcus aureus, Salmonella species (including Salmonellaenteritidis and Salmonella typhimurium), Clostridium botulinum,Pseudomonas aeruginosa, Campylobacter jejuni, Yersinia enterocolitica,Yersinia psudotuberculosis, Listeria monocyteogenes, Vibrio cholerae(both O1 and non-O1), Vibrio parahaemolyticus, Vibrio vulnificus,Clostridium perfringens, Bacillus cereus, Aeromonas hydrophila,Shigella, Streptococcus, Cryptosporidium, Giardia lamblia, Entamoebahistolytica, Cyclospora cayetanensis, Anisakis, Diphyllobothrium,Nanophyetus, Eustrongylides, Acanthamoeba, Ascaris lumbricoides,Trichuris trichiura, Legionella, fecal coliforms, non-fecal coliforms(such as Enterobacter, Klebsiella, Citrobacter), etc.

As discussed above, for a particular target, nanoparticles are selectedsuch that the target is at least 10-fold, at least 20-fold, at least30-fold, at least 50-fold, at least 100-fold, or at least 200-foldlarger than the nanoparticles.

Exemplary Samples

The present methods can be used to detect targets in a variety of sampletypes, including water samples (such as drinking water, water used forirrigation, and waste water); food; biological samples (such as bodilyfluids, pharmaceuticals, etc.); cosmetics; air samples; etc. In someembodiments, a sample is diluted, concentrated, suspended, and/ordissolved prior to, or during, the methods described herein. In someembodiments, a sample is at least partially separated prior to using itin the methods described herein, such as, for example, separating bloodfractions. In some embodiments, a buffering agent is added to a sampleprior to, or during, the methods described herein. In some suchembodiments, the buffering agent is added to bring and/or maintain thesample at a particular pH during at least a portion of the method. Oneskilled in the art can prepare a sample for use in the present methods,depending on the state of the sample (i.e., liquid, solid, gel, paste,etc.), the thickness of the sample, the density of the sample, the pH ofthe sample, the predicted microbial concentration in the sample, etc.

Exemplary Methods of Detecting Targets

In some embodiments, methods of determining whether or not a samplecomprises a target are provided. In some embodiments, the methodcomprises contacting the sample with a linker and nanoparticles anddetecting aggregation of the nanoparticles. In some embodiments,aggregation of the nanoparticles indicates the absence of the target inthe sample. In some embodiments, minimal or no aggregation of thenanoparticles indicates the presence of the target in the sample. Insome such embodiments, the linker is present at a lower concentrationthan the nanoparticles.

In some embodiments, the method comprises contacting the sample with alinker and nanoparticles and detecting aggregation of the nanoparticles.In some embodiments, aggregation of the nanoparticles indicates thepresence of the target in the sample. In some embodiments, minimal or noaggregation of the nanoparticles indicates the absence of the target inthe sample. In some such embodiments, the linker is present at an excessconcentration relative to the nanoparticles.

In some embodiments, the linker is added to a sample before thenanoparticles are added. In some such embodiments, if target is presentin the sample, the linker binds to the target. If no target is present,the linker remains in solution. In some embodiments, nanoparticles arethen added. If the linker is in solution, and not bound to the target,in some embodiments, the linker facilitates aggregation of thenanoparticles. If the linker is bound to target cells, in someembodiments, its ability to facilitate aggregation of the nanoparticlesis impaired and little or no aggregation occurs. Thus, in someembodiments, the absence of the target is indicated by aggregation ofthe nanoparticles.

In some embodiments, the linker is present at an excess concentrationrelative to the nanoparticles. In some such embodiments, if no target ispresent, the excess linker prevents aggregation of the nanoparticles. Insome embodiments, the linker is present at a concentration sufficient toprevent aggregation of the nanoparticles in the absence of target. Insome embodiments, if target is present, sufficient linker is bound tothe target such that the remaining linker in solution facilitatesaggregation of the nanoparticles. Thus, in some such embodiments, thepresence of the target is indicated by aggregation of the nanoparticles.

A sample to which linker and nanoparticles have been added is referredto, in some embodiments, as a “test sample.”

In some embodiments, linker is added at a concentration such that asmall number of targets will “soak up” most or all of the linker. Insome embodiments, for example, if a linker is a biotinylated antibody,the linker is added at a concentration of between about 0.1 μg/mL and100 μg/mL. In some embodiments, the linker is added at a concentrationof between about 0.1 μg/mL and about 50 μg/mL. In some embodiments, thelinker is added at a concentration of between about 0.1 μg/mL and about25 μg/mL. In some embodiments, the linker is added at a concentration ofbetween about 0.1 μg/mL and about 10 μg/mL. In some embodiments, thelinker is added at a concentration of between about 0.1 μg/mL and about3 μg/mL. In some embodiments, the linker is added at a concentration ofbetween about 0.5 μg/mL and about 3 μg/mL. An appropriate linkerconcentration can be determined by one skilled in the art, consideringsuch factors as the identity of the linker, the identity of the target,the number of binding sites for the first functionality on the target tobe detected, the efficiency of binding to the target, the predictednumber of targets in a sample, etc.

In some embodiments, linker is added at an excess concentration, suchthat a small number of targets will “soak up” some of the linker,leaving sufficient linker in solution to facilitate aggregation of thenanoparticles. In some such embodiments, for example, if a linker is abiotinylated antibody, the linker is added at a concentration of betweenabout 0.1 μg/mL and 100 μg/mL. In some embodiments, the linker is addedat a concentration of between about 1 μg/mL and about 100 μg/mL. In someembodiments, the linker is added at a concentration of between about 1μg/mL and about 50 μg/mL. In some embodiments, the linker is added at aconcentration of between about 1 μg/mL and about 25 μg/mL. In someembodiments, the linker is added at a concentration of between about 1μg/mL and about 10 μg/mL. In some embodiments, the linker is added at aconcentration of between about 2 μg/mL and about 10 μg/mL. In someembodiments, the linker is added at a concentration of between about 2μg/mL and about 5 μg/mL. An appropriate linker concentration can bedetermined by one skilled in the art, considering such factors as theidentity of the linker, the identity of the target, the number ofbinding sites for the first functionality on the target to be detected,the efficiency of binding to the target, the predicted number of targetsin a sample, etc.

In some embodiments, nanoparticles are added at a concentration suchthat aggregation of the nanoparticles by the available linker isdetectable above the background of non-aggregated nanoparticles. As anonlimiting example, if gold nanoparticles between 10 nm and 15 nm insize are used to detect, e.g., E. coli, a concentration of nanoparticlesbetween 1 nM to 20 nM, 5 nM to 15 nM, or between 5 nM and 10 nM may beused. An appropriate nanoparticle concentration can be determined by oneskilled in the art, considering such factors as the type ofnanoparticle, the identity of the linker, the identities of the secondand third functionalities, the efficiency of binding of the second andthird functionalities, the number of binding sites for the nanoparticleson the linkers, the concentration of linker in the test sample, etc. Theconcentration should be selected such that there is an observabledifference between aggregated and non-aggregated nanoparticles.

In some embodiments, detection of aggregation is carried out bydetecting a change in at least one characteristic of the test sample. Insome embodiments, the change in at least one characteristic of the testsample is determined as a change from time 0, immediately after thenanoparticles are added, to a time T after incubation of the testsample. In some embodiments, time T is 5 minutes, 10 minutes, 15minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45minutes, 50 minutes, 55 minutes, 60 minutes, 70 minutes, 80 minutes, 90minutes, 100 minutes, or 120 minutes. Thus, in some embodiments, acharacteristic of the sample at time 0 is compared to the samecharacteristic of the sample at time T.

In some embodiments, the change in at least one characteristic of thetest sample is determined by comparing the at least one characteristicof the test sample to one or more standards. In some embodiments, thestandard is a control sample. In some embodiments, a control samplecomprises the same linker and nanoparticles as the test sample, but doesnot contain the target. In some embodiments, the control sample hasundergone the same processing as the test sample, although theprocessing of the control sample may or may not have occurredcontemporaneously with the processing of the test sample.

In some embodiments, a standard is a representation of at least onecharacteristic of a test sample or a control sample. Such arepresentation may be of a sample that comprises the target, or a samplethat lacks the target. In some embodiments, at least one characteristicof a test sample is compared to both a standard that represents the atleast one characteristic in a sample that comprises the target, and astandard that represents the at least one characteristic in a samplethat lacks the target.

Various characteristics of a test sample may be used to determinewhether the nanoparticles have aggregated. Nonlimiting exemplarycharacteristics include color, UV-visible spectrum, UV-visible peakwavelength, and absorbance.

In some embodiments, aggregation of nanoparticles causes a change in atest sample that is visible to the naked eye. In some embodiments,aggregation of nanoparticles causes a color change in a test sample. Insome embodiments, for example, when gold nanoparticles are used, a testsample in which the nanoparticles have not aggregated is red in color,while a test sample in which the nanoparticles have aggregated is purplein color. In some embodiments, the peak wavelength of a test sample inwhich the nanoparticles (such as gold nanoparticles) have not aggregatedis about 530 nm, while the peak wavelength of a test sample in which thenanoparticles have aggregated is about 550 nm to 560 nm, with increasedabsorption at wavelengths above 600 nm.

In some embodiments, the presence of aggregation in a test sampleindicates that the target is not present at levels detectable by thatparticular assay. Thus, in some embodiments, if gold nanoparticles arebeing used in the method, and the test sample turns purplish over timeand/or the peak UV-visible wavelength shifts from about 530 nm to 550 nmor longer, the sample does not contain a detectable amount of thetarget.

In some embodiments, the absence of aggregation in a test sampleindicates that the target is present. Thus, in some embodiments, if goldnanoparticles are being used in the method, and the test sample remainsred over time and/or the peak UV-visible wavelength remains at about 530nm, the sample contains the target.

In some embodiments, the presence of aggregation in a test sampleindicates that the target is present. Thus, in some embodiments, if goldnanoparticles are being used with an excess amount of antibody in themethod, and the test sample turns purplish over time and/or the peakUV-visible wavelength shifts from about 530 nm to 550 nm or longer, thesample contains a detectable amount of the target.

In some embodiments, the absence of aggregation in a test sampleindicates that the target is not present at levels detectable by thatparticular assay. Thus, in some embodiments, if gold nanoparticles arebeing used with an excess amount of antibody in the method, and the testsample remains red over time and/or the peak UV-visible wavelengthremains at about 530 nm, the sample does not contain the target.

In some embodiments, the absorbance of the sample at a particularwavelength is detected with a colorimeter in order to detectaggregation. In some embodiments, a change in the absorbance at aparticular wavelength indicates a change in the amount of aggregation ina sample, and the presence or absence of the target. In someembodiments, for example, for a system using gold nanoparticles and alimiting amount of an antibody to the target, if the absorbance at 530nm is detected, and the absorbance decreases after addition of thesample, the sample does not contain a detectable amount of the target(i.e., aggregation occurs, and the peak wavelength shifts to 550 nm orlonger, reducing the absorbance at 530 nm). In other embodiments, forexample, for a system using gold nanoparticles and an excess amount ofan antibody to the target, if the absorbance at 530 nm is detected, andthe absorbance decreases after addition of the sample, the samplecontains a detectable amount of the target (i.e., aggregation occurs,and the peak wavelength shifts to 550 nm or longer, reducing theabsorbance at 530 nm).

In some embodiments, the color of the sample is determined using ahistogram of a color image of the sample. In some embodiments, the colorof the sample is determined using a Turboscan.

Exemplary Standards

In some embodiments, at least one characteristic of a test sample iscompared to at least one characteristic of a standard. In someembodiments, a standard is a control sample that has been treated underthe same conditions, and comprises the same linker and nanoparticles, asthe test sample, but lacks the target. One skilled in the art caninclude a suitable control sample in a method if desired.

In some embodiments, a standard is a representation of at least onecharacteristic of a test sample. For example, in some embodiments, ifthe at least one characteristic is the color of the test sample, astandard may be a representation of one or more colors to which the testsample can be compared. In some embodiments, if the at least onecharacteristic is a UV-visible spectrum of the test sample, a standardmay be a representation of a UV-visible spectrum that would be expectedfrom a test sample comprising the target and/or a representation of aUV-visible spectrum that would be expected from a test sample that doesnot comprise the target. One skilled in the art can make and use asuitable standard according to the characteristic to be considered andthe particular method employed.

Exemplary Kits

In some embodiments, kits are provided. In some embodiments, a kitcomprises a linker and nanoparticles. In some embodiments, a kit furthercomprises at least one standard. Nonlimiting exemplary linkers,nanoparticles, and standards are described herein.

The following examples are offered by way of illustration and are notintended to limit the invention in any way.

EXAMPLES Example 1 Materials and Methods

Deionized water, filtered at 18 Ω·cm of resistivity, was used in allexperiments. Most chemicals (hydrogen tetrachloroaurate, trisodiumcitrate, streptavidin, bovine serum albumin (BSA), borate buffer,phosphate buffered saline (PBS), HEPES buffer, glycerine and NaOH) werepurchased from Fisher Scientific (Pittsburgh, Pa., USA) and used asreceived unless indicated otherwise. Biotin was purchased fromSigma-Aldrich (St. Louis, Mo., USA) and anti-tissue/cell preparation ofE. coli polyclonal rabbit IgG antibody (pAb) (GTX13626) and biotinylatedantibody (GTX40640) were from Genetex Inc. (Irvine, Calif., USA). DH5αE. coli was cultured in LB plates then gathered and diluted in PBS toobtain a 1×10¹⁰ CFU/mL suspension, which was then diluted as needed.

Synthesis of Gold Nanoparticles (AuNPs)

AuNPs (13-nm average diameter) were prepared by adding 10% v/v oftrisodium citrate to a boiling solution of 1 mM hydrogentetrachloroaurate. After the color of the solution turned wine red, thecitrate-stabilized AuNPs were stirred until the solution cooled down toroom temperature.

Functionalizing AuNPs with Antibody

AuNPs were functionalized with antibody by charge adsorption as follows.500 μL of freshly prepared colloidal AuNPs was mixed with 400 μL boratebuffer (pH 7.4), and then 100 μL of pAb solution (400 μg/mL in PBS) wasadded. After 10 min of incubation, the mixture was centrifuged andwashed with borate buffer (pH 9.5) twice to remove unbound pAb.Centrifuged pAb-conjugated AuNPs were stored in 500 μL PBS/0.1% w/vsodium azide.

Functionalizing AuNPs with Streptavidin

AuNPs were functionalized with streptavidin by charge adsorption asfollows. The streptavidin solution was prepared in borate buffer (pH7.4) at 50 μg/mL. 400 μL of the streptavidin solution was mixed with 600μL of freshly prepared colloidal AuNPs (estimated to be at aconcentration of about 8 nM). Following 30 min of incubation, thestreptavidin-coated AuNPs were centrifuged, the supernatant was removed,the streptavidin-coated AuNPs were washed several times, and thenresuspended in 600 μL of PBS/0.1% bovine serum albumin

The biotin-dependence of the aggregation of streptavidin functionalizedAuNPs in the presence of biotinylated antibodies was confirmed asfollows. 100 μL of 10 μg/mL biotin was added to 200 μL ofstreptavidin-coated AuNPs. After 15 minutes, 100 μL of biotinylated pAbwas added to the biotin-treated AuNPs. No visible color change wasobserved, suggesting that the aggregation of the AuNPs is dependent onthe binding of the streptavidin on the AuNPs to the biotinylatedantibodies.

UV-Vis Absorption Spectra

Absorption spectra were measured using a UV-vis spectrophotometer(UV-1601PC, Shimadzu, Columbia, Md., USA). For sample preparation, PBSwas added to each reaction sample to a final volume of 1 mL.

Example 2 On-Target Detection Resulted in No Visible Color Change

To investigate the effect of attaching AuNPs on E. coli on the overallcolor of the sample solution, 200 μL of pAb-conjugated AuNPs were addedto 200 μL of various concentrations of E. coli in PBS, for total E. coliconcentrations of 10¹ to 10⁹ CFU/400 μL. The samples were then incubatedat room temperature for one hour. The attachment of AuNPs on E. coli wasverified by examining the color of sediment obtained by lightlycentrifuging the solution at 3000 rpm for 10 min.

The results of that experiment are shown in FIG. 1. Even at the highestconcentrations of E. coli tested, no visually distinguishable colorchange was observed. See FIG. 1A. Following centrifugation, however, thepresence of a dark-red sediment confirmed that the AuNPs were bound tothe E. coli. See FIG. 1B. However, the color of the supernatant remainedunchanged. Compare tube on left (no E. coli control) with tube on theright (10⁹ CFU/400 μL E. coli). These results suggest that only a smallfraction of the AuNPs in the sample is bound to the cell surface, andthat this fraction is insufficient to cause a visible color change inthe system.

Example 3 On-Target Detection with Lower Concentration of AuNPs Resultedin No Visible Color Change

Next, the effect of lowering the initial AuNPs concentration wasinvestigated. The AuNPs were diluted with PBS to a concentration of 25%of its original level. In addition, in this experiment, biotinylatedantibody was first bound to the E. coli, and thenstreptavidin-functionalized AuNPs were added. After binding of thebiotinylated antibodies to E. coli, the cells were washed multiple timesto remove unbound antibodies.

For direct visual comparison of the sample color, all tests wereperformed under the same conditions, keeping sample volumes and particleconcentrations the same. Samples were diluted in PBS to 200 μL, and then200 μL of streptavidin-coated AuNPs was added to bind the AuNPs to pAb.For the control, the same procedure was followed without thebiotinlyated pAb such that AuNPs would not specifically bind to the cellsurface.

The results of that experiment are shown in FIG. 2. The binding of AuNPsto E. coli (10⁹ CFU/400 μL) at both high and low concentrations of AuNPswas verified by examining the color of the centrifuged sediment. SeeFIG. 2. FIG. 2A shows samples containing higher a higher concentrationof AuNPs (100% concentration). FIG. 2B shows samples containing a lowerconcentration of AuNPs (25% concentration). Although the color of thesamples containing a lower AuNPs concentration was lighter, there wasstill no distinguishable change in the sample color upon binding ofAuNPs to E. coli. Compare sample on left (with biotinylated antibody) tosample of right (without biotinylated antibody) in FIG. 2B.

The above experiments further indicate that the binding of AuNPsdirectly to bacteria does not change the sample color sufficiently foron-target visual detection of bacteria. Without being bound by anyparticular theory, this result may be due to one or more of thefollowing reasons. First, it may be that only a small fraction of thetotal AuNPs binds to the bacteria, even at a low initial AuNPconcentration. Second, considering that AuNPs bind to bacteria at theantigen sites, uniform covering of AuNPs on the cell surface may beunlikely because antigen sites are not equally spaced and/or therelatively large AuNPs, compared to inter-antigen spacing, occludeseveral antigen sites on the cell surface. See FIG. 3A. Third, it may bethat monolayered AuNP binding to the cell surface cannot produce aplasmonic effect strong enough for visual indication as wouldthree-dimensional clusters of AuNPs that occur off-target. See FIG. 3B.

Example 4 Observable Color Change Using Off-Target Detection

Because on-target binding of AuNPs to cells did not result in a visiblecolor change in the above experiments, the potential of detecting cellsusing off-target aggregation of AuNPs was investigated. According to themanufacturer, the biotinylated antibody is labeled with 7 to 10 biotinsper unit. Therefore, one free antibody should be able to crosslinkmultiple strepavidin-coated AuNPs and facilitate color-changingaggregation of AuNPs. An unaggregated solution of AuNPs is red, and thecolor shifts to purple as the AuNPs aggregate. The ability of thebiotinylated antibody to cause the color shift was confirmed byincubating the antibody with streptavidin-coated AuNPs, with and withoutexcess biotin. Data not shown. In the absence of excess biotin, additionof biotinylated antibody to streptavidin-coated AuNPs results in a colorchange from red to purple. When excess biotin is added, which blocks thebiotin binding sites on the streptavidin-coated AuNPs and prevents thebiotinylated antibody from crosslinking the AuNPs, the color change doesnot occur.

To test the off-target detection of cells, 100 μL of E. coli suspendedin PBS at 10², 10⁴, 10⁶, and 10⁸ CFU/mL was mixed with 100 μL of 10μg/mL biotin-conjugated antibody in PBS (resulting in E. coliconcentrations of 10, 10², 10³, and 10⁴ CFU/mL and 5 μg/mL antibody).The mixture was incubated for 30 minutes with vigorous stirring. Afterthe incubation, 200 μL of streptavidin-coated AuNPs was added and thecolor change was monitored as a function of time.

The results of that experiment are shown in FIG. 4. A visible colorchange from red to purple occurs within about 15 min. See FIG. 4A. Atone hour, a more pronounced color change is observed. See FIG. 4B. Whilethe color change is not directly proportional to the E. coliconcentration, a shift in the UV-VIS peak absorbance is evident when E.coli are present. See FIG. 4C. Accordingly, at the very least, thisoff-target method produced a yes/no indication of the presence ofbacteria in this experiment, even at E. coli concentrations as low as 10CFU/mL.

Similar experiments were carried out with Pseudomonas aeruginosa andSalmonella typhimurium, demonstrating that the off-target detectionmethod can detect a range of targets.

Example 5 Characteristics of AuNP Aggregation

In order to examine the color-changing characteristics of AuNPaggregation as a function of target concentration, an assay system wasdesigned using streptavidin-coated AuNPs and biotinylated BSA.

Biotinylated BSA (Sigma-Aldrich; 0.5 μg in 100 μl) was mixed with 200 μlstreptavidin-coated AuNPs made as described in Example 1, in a totalvolume of 400 μl (brought up to volume with PBS). The mixture wasincubated at room temperature (˜10° C. to 30° C.). The absorptionspectrum of the mixture was taken at 0 hour, 1 hour, 2 hours, and 3hours to determine the absorbance and peak wavelength changes of themixture over time, as the streptavidin-coated AuNPs bind to thebiotinylated BSA. As shown in FIG. 5, the peak absorbance of the mixturedecreased with time, while the peak absorption wavelength increased. Atthe same time that the peak absorbance decreased, the absorbance athigher wavelengths increased (see large arrows in FIG. 5). These changesindicate that the streptavidin-coated AuNPs were aggregating in thepresence of the biotinylated BSA.

The relationship between the concentration of biotinylated BSA and thechange in the peak absorbance over time was then investigated.Increasing amounts of biotinylated BSA in 100 μl (0.5, 1, 1.5, 2, 2.5,and 3 ng/100 μl) were incubated with 200 μl of streptavidin-coated AuNPsin a total volume of 400 μl (brought up to volume with PBS), and themixture was incubated at room temperature. The absorption spectrum ofeach mixture was taken at 30 minutes, 1 hour, 2 hours, and 3 hours, andthe Δλmax measured from each time point to the next, at eachconcentration of biotinylated BSA. The results of that experiment areshown in FIG. 6A. The greatest peak wavelength changes over timeoccurred at the lower amounts, or concentrations, of biotinylated BSA.At the lowest concentration of biotinylated BSA (e.g., 0.1 μg in thisexperiment), there is insufficient biotinylated BSA to aggregate theAuNPs, so they remain in solution with no wavelength change over time.At the “middle” concentrations of biotinylated BSA (e.g., 0.5 μg and 1μg in this experiment), there is sufficient biotinylated BSA toaggregate the AuNPs, resulting in peak wavelength changes over time. Athigher concentrations of biotinylated BSA (e.g., 1.5 μg and greater inthis experiment), there is excess biotinylated BSA and aggregation doesnot occur because there is sufficient biotinylated BSA to coat eachAuNP. These regions are shown in FIG. 6B (lowest concentrations=RegionA; “middle” concentrations=Region B; highest concentrations=Region C)and illustrated in FIG. 6C.The effect of free streptavidin on the colorchange observed in FIG. 6B was determined. A schematic representation ofthe assay is shown in FIG. 7. A similar experiment as described abovefor FIG. 6 was carried out, but in the presence of 0 μg streptavidin, 1μg streptavidin, or 10 μg streptavidin. Free streptavidin was incubatedwith the biotinylated BSA prior to addition of the AuNPs. Each mixturewas incubated at room temperature for 2 hours before the absorptionspectrum was taken. The Δλmax was measured from each time point to thenext, at each concentration of biotinylated BSA. The results of thatexperiment are shown in FIG. 8. The addition of free streptavidin causedthe greatest Δλmax to shift to a higher concentration of biotinylatedBSA. In other words, more biotinylated BSA was required to aggregate thestreptavidin-coated AuNPs in the presence of free streptavidin. Compare,e.g., FIG. 8 inset to FIG. 6B.

A similar experiment was carried out using various concentrations ofbiotinylated antibody, preincubated with 10 μg, 1 μg, or 10 μg freestreptavidin, and then mixed with 200 μl of streptavidin-coated AuNPs,in a total volume of 400 μl (brought up to volume with PBS). The resultsof that experiment are shown in FIG. 9. Briefly, the antibodyconcentration at which the greatest Δλmax shifted from 2 μg biotinylatedantibody (in the presence of 0 or 1 μg streptavidin) to 3 to 4 μgantibody (in the presence of 10 μg streptavidin).

Example 6 Sensitivity of Off-Target Microorganism Detection Using AuNPs

An exemplary method and model for off-target microbial detection usingAuNPs is shown in FIG. 10. Briefly, in a first step, a sample that mayor may not comprise a microorganism is incubated with biotinylatedantibodies that bind, for example, to a marker on the surface of themicroorganism. If microorganisms are present, the biotinylatedantibodies bind to the surface of the microorganisms. In a second step,strepatavidin-coated AuNPs are added to the sample. If there are nomicroorganisms in the sample, the biotinylated antibody cross-links thebiotinylated AuNPs and large-scale aggregates form, causing a visualcolor change in the sample from red to purple. If microorganisms arepresent, the streptavidin-coated AuNPs bind to the biotinylatedantibodies on the surface of the microorganisms, preventing large-scaleaggregation of the AuNPs and the color change.

In order to determine the potential sensitivity of the off-targetdetection assay illustrated in FIG. 10, the assay was carried out usinga range of biotinylated antibody concentrations (0, 0.5, 1, 2, 3, 4, and5 μg in 100 μl) and a range of bacterial concentrations (0, 10², 10⁴,and 10⁶ CFUs in 100 μl), with 200 μl streptavidin-coated AuNPs (totalfinal volume of 400 μl). The mixtures were incubated at room temperaturefor 2 hours.

The results of that experiment are shown in FIG. 11. In this experiment,it was demonstrated that two different color change phases can beobserved. At 1 μg biotinylated antibody, indicated by the first red box,the streptavidin-coated AuNPs are aggregated in the absence of bacteria(0 CFU), and the color of the mixture is purple. This aggregation occursbecause the biotinylated antibody concentration to streptavidin-coatedAuNP concentration is such that there is less biotinylated antibody thanstreptavidin-coated AuNP, allowing multiple streptavidin-coated AuNPs tobind to each biotinylated antibody, which results in aggregation. Withjust 100 CFUs of bacteria present, the biotinylated antibodies are boundto the surface of the bacteria and no longer aggregate thestreptavidin-coated AuNPs, resulting in a red color. At 4 μgbiotinylated antibody, indicated by the second red box, the AuNPs arenot aggregated in the absence of bacteria (0 CFU) because there is anexcess of biotinylated antibody (see, e.g., FIG. 6C, “Region C”). With10⁴ CFUs of bacteria present, however, aggregation begins to occurbecause much of the biotinylated antibody is “soaked up” by thebacteria, leaving enough biotinylated antibody in solution to aggregatethe streptavidin-coated AuNPs.

Thus, the present system can be fine-tuned to a very high level ofsensitivity for a particular threshold of microbial contamination. Forexample, in some embodiments, for testing a sample in which little or nomicrobial contamination can be tolerated, such as food orpharmaceuticals, a lower amount of biotinylated antibody is used, andthe system will be purple in the absence of contamination, and turn redat very low levels of contamination. In some embodiments, for testing asample in which higher levels of microbial contamination are tolerated,or if the sample has been concentrated, higher amounts of biotinylatedantibodies are used, and the system will be red in the absence ofcontamination, and turn purple at higher levels of contamination.

The foregoing description is considered as illustrative only and is notintended to limit the claimed invention. Numerous modifications andchanges may readily occur to those skilled in the art. The invention isnot limited to the exact construction and operation shown and described,and accordingly, all suitable modifications and equivalents areconsidered to fall within the scope of the invention.

1. A method of determining whether a sample comprises a target,comprising: a) contacting the sample with a linker, wherein the linkercomprises a first functionality and a plurality of secondfunctionalities, wherein the first functionality is capable of bindingto the target, and wherein each of the plurality of secondfunctionalities is capable of binding to a third functionality; b)contacting the sample from (a) with a plurality of nanoparticles,wherein each of the plurality of nanoparticles comprises a thirdfunctionality that is capable of binding to the second functionality,and wherein the linker is added in step (a) at a concentration less thanthe concentration of nanoparticles and sufficient to aggregate thenanoparticles added in step (b) in the absence of target; and c)detecting nanoparticle aggregation in the sample from (b), wherein theabsence of nanoparticle aggregation indicates that the sample comprisesthe target.
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. The method ofclaim 1, wherein the linker is an antibody and the first functionalityis the antigen binding region of the antibody.
 6. The method of claim 5,wherein the second functionality is biotin and the third functionalityis streptavidin.
 7. The method of claim 1, wherein the nanoparticles areselected from gold nanoparticles, silver nanoparticles, platinumnanoparticles, magnetite nanoparticles, gold/iron alloy nanoparticles,and latex nanoparticles.
 8. The method of claim 1, wherein the target isselected from prokaryotic cells, eukaryotic cells, and parasites.
 9. Themethod of claim 1, wherein detecting nanoparticle aggregation comprisesdetermining at least one characteristic selected from sample color,UV-VIS spectrum, UV-VIS peak wavelength, and absorbance.
 10. The methodof claim 9, wherein the at least one characteristic of the sample from(b) is compared to at least one characteristic of a standard. 11.(canceled)
 12. (canceled)
 13. A method of determining whether a samplecomprises a target, comprising: a) contacting the sample with a linker,wherein the linker comprises a first functionality and a plurality ofsecond functionalities, wherein the first functionality is capable ofbinding to the target, and wherein each of the plurality of secondfunctionalities is capable of binding to a third functionality; b)contacting the sample from (a) with a plurality of nanoparticles,wherein each of the plurality of nanoparticles comprises a thirdfunctionality that is capable of binding to the second functionality,and wherein the linker is added in step (a) at a concentration just inexcess relative to the concentration of the nanoparticles added in step(b) and the nanoparticles do not aggregate in the absence of target; andc) detecting nanoparticle aggregation in the sample from (b), whereinthe presence of nanoparticle aggregation indicates that the samplecomprises the target.
 14. (canceled)
 15. (canceled)
 16. (canceled) 17.The method of claim 13, wherein the linker is an antibody and the firstfunctionality is the antigen binding region of the antibody.
 18. Themethod of claim 17, wherein the second functionality is biotin and thethird functionality is streptavidin.
 19. The method of claim 13, whereinthe nanoparticles are selected from gold nanoparticles, silvernanoparticles, platinum nanoparticles, magnetite nanoparticles,gold/iron alloy nanoparticles, and latex nanoparticles.
 20. The methodof claim 13, wherein the target is selected from prokaryotic cells,eukaryotic cells, and parasites.
 21. (canceled)
 22. The method of claim13, wherein detecting nanoparticle aggregation comprises determining atleast one characteristic selected from sample color, UV-VIS spectrum,UV-VIS peak wavelength, and absorbance.
 23. The method of claim 22,wherein the at least one characteristic of the sample from (b) iscompared to at least one characteristic of a standard.
 24. (canceled)25. (canceled)
 26. A kit for determining whether a sample comprises atarget, comprising (i) a linker, wherein the linker comprises a firstfunctionality and a plurality of second functionalities, wherein thefirst functionality is capable of binding to the target, and whereineach of the plurality of second functionalities is capable of binding toa nanoparticle; (ii) a plurality of nanoparticles, wherein each of theplurality of nanoparticles comprises a third functionality that iscapable of binding to the second functionality; and (iii) a standard,wherein the standard is a representation of at least one characteristicof a control reaction that comprises the linker and the plurality ofnanoparticles, but not the target.
 27. (canceled)
 28. (canceled) 29.(canceled)
 30. The kit of claim 26, wherein the linker is an antibodyand the first functionality is the antigen binding region of theantibody.
 31. The kit of claim 30, wherein the second functionality isbiotin and the third functionality is streptavidin.
 32. The kit of claim26, wherein the nanoparticles are selected from gold nanoparticles,silver nanoparticles, platinum nanoparticles, magnetite nanoparticles,gold/iron alloy nanoparticles, and latex nanoparticles.
 33. (canceled)34. (canceled)
 35. The kit of claim 26, wherein the at least onecharacteristic is selected from sample color, UV-VIS spectrum, UV-VISpeak wavelength, and absorbance.